When I pour medium from the CELLine, I sometimes have a drop of medium left on the outside of the neck, what can I do to stop this from happening?

When pouring medium from the CELLine it is recommended that the flask be held with the bottom of the flask in the palm. This provides adequate neck pouring angle and prevents accumulation of the medium on lip of neck after pouring. The angle of the neck on the CELLine is required to prevent air locks in the nutrient reservoir and thus pouring is best done with flask upside down in palm of hand.

After incubation, I notice that the outside of the CELLine is wet, why is this?

If medium is placed in flask that is not pre-warmed, there will be considerable condensation accumulation on the outside of the CELLine. Due to the large volume of medium contained in the CELLine, this condensation can be significant. The condensate takes time to evaporate in the humidified incubator. Test color of liquid by blotting with white paper, if there is no coloration, the liquid is water and due to condensate.

Can I place more than the recommended volumes into the cell compartment?

The protocol recommends a working volume of 5 ml (CL 350), and 15 ml (CL or AD 1000) for the CELLine products. This assures that volume in the cell compartment never exceeds bursting threshold for the membrane even with osmotic flux of water into the cell compartment over an extended period. The membrane is fragile but compliant and will distend significant distance when wet. Increased cell compartment volumes up to 1.3 times above recommendation are not problematic.

Will I be able to recover all of my cells from the cell compartment?

When working with cells growing in suspension in CELLine classic, the recovery of cells from the cell compartment should be nearly 100%. Suspension cell types have not been observed to form aggregates and are readily disbursed with gentle pipetting. Following pipetting the cells are easily recovered. An additional rinse of clean medium may be used to further assure complete cell recovery if required.
Other cells types may form cellular aggregates or attach to the bottom of the cell compartment. In these cases, cell recovery may require the use of a dissociating agent to separate cells and to aid in their recovery.

When working with anchorage-dependent cells in CELLine adhere, cell are attached to the PET ilay matrix. Depending on the individual cell type, sometimes recovery of the cells can be achived mechniaclly by pipetting up and down, but in most of the cases a complete recovery involves the addition of a dissociating agent to the cell compartment.

Can I change nutrient medium by tracking color change of the nutrient medium as I do in my other cultures?

It is not recommended. Cell growth is dependent on the diffusion gradients present between the cell compartment and nutrient medium compartment. The greater the gradient, the larger the flux of soluble metabolic substrates that is available for cell metabolism. The medium color change is not an accurate assessment of nutrient and waste status in the CELLine due to the ability of the cell compartment to balance pH directly with the incubator atmosphere. Nutrient medium will become more yellowish during culture but will not take on the characteristic color associated with spent medium in traditional flasks. Careful tracking of cell numbers within the cell compartment and medium exchange rate can be done to determine optimum conditions for your cell type.

7.

When I harvest from the cell compartment, I always have a greater volume than what I inoculated why is this happening?

Osmotic gradients across the upper semi-permeable membrane will drive water through the membrane. If a protein gradient is present across the semi-permeable membrane such as when no serum is used in the nutrient compartment, water is driven into the cell compartment. Because small solutes will move across the membrane also, this change in volume only affects colloid protein concentrations.

This is the reason for the recommended use of 15% serum in the cell compartment when no serum is used in the nutrient medium, as it assures that serum concentrations with in the cell compartment do not become excessively diluted with continued culture.

8.

The cell compartment volume in my CELLine is less than what I inoculated, where is the volume going?

If the CELLine is used in a non humidified incubator or warm room, evaporative losses from the cell compartment can lead to reduced volumes. The CELLine is intended to be used in a standard humidified incubator. If required it may be worth evaluating restricting the bottom gas vents to reduce evaporation. This should be done experimentally to determine the balance between evaporative loss and adequate gas exchange.

I notice that the distribution of cells in the cell compartment sometimes is not even across the bottom of the cell compartment, should I mix the cell compartment to provide a more even distribution?

I have viewed my cells in the CELLine classic but when I tried to view them again after several days of culture I was not able to focus on them, what has changed?

How strong is the semi-permeable membrane?

The upper semi-permeable membrane is only 8 µm thick (dry). The membrane is delicate but easily withstands normal handling. Shaking or banging of flask against hand or surface can lead to membrane failure if the cell compartment has liquid in it. This places significant stress on the membrane due to the rapid displacement of the liquid present in the cell compartment.

16.

Why is it recommended to wet the membrane before placing cells into the cell compartment?

It is important to wet the membrane prior to inoculation to assure that the membrane is compliant. The wet membrane is compliant and capable of distension. The dry membrane is more susceptible to tensile stress due to volume changes. The air trapped in the cell compartment can not be removed until the membrane is wet and liquid is added into the cell compartment. The dry membrane is stressed significantly if volume is added directly into the cell compartment prior to wetting of membrane.

Does the semi-permeable membrane become clogged with use?

Performance of the CELLine devices does not decrease with time of culture. This indicates that solute transfer across the membrane does not decrease significantly during culture and there is thus no significant clogging or fouling of the membrane.

Are there different membranes available for the CELLine?

Currently, only a 10,000 MWCO membrane is available.

24.

Can I culture leukemic cell lines in the CELLine?

Lymphoblastic cells grow very well in the CELLine classic. Cell concentrations of certain lymphoblastic cells can reach nearly twice that achieved for hybridoma cells. Some cell lines may be dependent upon the use of serum on both sides of the semi-permeable membrane and this can be readily examined.

Do you recommend the use of a high glucose containing medium in the CELLine?

Most protocols were developed using standard RPMI-1640 medium. Some customers and others who use hollow fiber bioreactors do use richer mediums. A slight performance increase may be obtained with richer media, however, this is dependent upon cell line and should be evaluated experimentally. In general excellent results are obtained in medium which is currently used to culture the cell line in static flasks.

Will serum free medium work in the CELLine?

Yes many customers report excellent results using serum free medium. The serum free medium is placed on both sides of the semi-permeable membrane in most cases. Importantly, the use of serum free medium may no longer be necessary when the CELLine is used. As the secreted protein is recovered at high concentration from the CELLine, it is no longer necessary to concentrate culture supernatant to recover antibody. This eliminates much of the interference associated with serum protein during purification. At antibody concentrations in excess of mg/ml, cell compartment supernatant can be applied directly to an affinity column.